REVERSE GENETICS AND ITS APPLICATIONS :

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What is reverse genetics? :

What is reverse genetics? A methodology for studying the function of each gene in genomes
Is a tool to investigate the impact of induced variation in a specific gene on the organism
Rescue of virus entirely from transfecting cloned cDNA plasmids encoding the viral components
positional cloning

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Recovery of genetically manipulated DNA viruses achived first time for SV- 40 by transfecting mutated cloned cDNA to produce defective mutant viruses (Goff and Berg ,cell,1976)
Rescue of large DNA containing herpes virus utilizing h.recombination leading to construction of herpes virus variants(Post and Roizman,cell,1981)
Live virus vector era began with development of vaccinia virus vector(Smith,Mackett and Moss, 1983)

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RG pox virus
Construction of chimeric vaccinia virus-herpesvirus plasmids, rescue of rec. virus
Vaccinia genomic DNA is digested with Hind |||
Clone it in the vector pBR-322to get pDP3
Obtain a TK gene segment with ends having Bam H |
Clone it in Bam H | site of pDP3
Check the orientation and no copies of TK by digestion with Sst | and fragment analysis
When these plasmids cotransfected with helper virus rec virus with TK gene generated Panicali and Paoleti ,pnas,1982

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Gene manipulation and rescue of negative sense RNA viruses was hindered for reasons like……
Genomic RNA of these viruses is not infectious it requires RNPcomplex
Infectious particles deliver their own RNA dependent RNA polymerase to start virus replication

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In 1994 RG for Rabies virus entirely from cDNA was developed by Schnell,Mebatason and conzellman
This method was quickly used to develop RG system for other NNSV like ……
Human respiratory syncytial virus
human parainfluenza virus
NCD ,measels,VSV etc

PBRG of NNSV :

PBRG of NNSV Transfect cell with plasmid encoding………
Full length genomic /antigenomic RNA
Major proteins for transcription and replication
Drive plasmid expression by phase T7 polymerase
T7 polymerase made available by
1)cotransfection with plasmid encoding it
2)with helper vaccinia virus expressing it
3)using the cell lines which endogenously express it

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Generation of influenza A virus entirely from cloned c DNA
Plasmids constructed for each of 8 segments of v RNA as cDNA in between RNA pol 1 promoter and terminator seq
cDNA of each genes of viral proteins are cloned in diff eukaryotic expression plasmids
Transfection of 293T human embrioyanic kidney cell line with different amounts of expression plasmids and RNA pol 1plasmids –total 17 diff plasmids used
(Neumann,Watanabe etal,PNAS,1999)

Potential Applications of RG of viruses :

Potential Applications of RG of viruses Structure –function study of individual viral genes…not by isolation in different system
(reductionist approach)
In viral infectious system ..
their interaction with host cell
To study molecular basis of pathogenicity of virus
ex:- F protein cleavage by host protease determine the
virulence of NDV(Ben &Olav J.viro,1999)
RG provides direct evidence for correlation between
HA cleavability and virulence of Avian Influenza A
virus(Tisuke &Yoshihiro,J.Viro,1994)

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As live virus vaccines….
with deletion or alteration of virulent sequences
Genetically engineered live attenuated Influenza A virus vaccine candidates(Neil&Peggy,J.vir,1997)
Recombinant NDV expressing VP2 of IBD protects against both NDV and IBD(Zhuhui ,Subbiah and Abdul,J.Viro,2004)
As viral vectors expressing foreign protein for vectored vaccines, gene therapy ,cancer treatment manufacture of biologicals
Speculation of genetic vaccines using defective viral RNA genomes that replicate but not spread to neighboring cells

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Summary RG is a marked forward leap in understanding molecular biology and pathogenesis viruses and development of effective vaccines against many viral disease of animals and birds
Development of effective vaccine for avian influenza to prevent the pandemics
RG has expanded the horizons of recombinant DNA technology such as effective viral vaccine ,vectors ,biologicals and there are efforts to make further refinements in this new field